NOTICE: You are viewing a page of the openwetware wiki. Our "dewikify" feature makes a wiki page appear as a normal web page. On September 1st 2017, this feature will GO AWAY and this URL will redirect to the source URL on our wiki. We're sorry for the inconvenience.

in progress, this protocol hasn't worked yet; use at your own risk!!!



This is a protocol to anneal and ligate oligo's to construct short DNA fragments for cloning (~100 bp)



  1. Resuspend primers.
  2. Kinase treat each oligo using the following reaction mix for each primer.
    • 2 μL primer (5 μM final concentration)
    • 1 μL 10X T4 DNA ligase buffer
    • 6.5 μL H2O
    • 0.5 μL T4 polynucleotide kinase
  3. Incubate at 37 °C for 1 hr 30 mins
  4. Heat kill at 65 °C for 20 mins
  5. Mix all 10μL of kinase treated oligo with its pair to make duplex oligos.
  6. Heat to 70-75 °C and cool to anneal oligo pairs.
  7. Mix all 20 μL of each duplex together in one tube.
  8. Make up ligation reaction
    • 1 μL of the oligo mix
    • 50 ng digested vector backbone
      • The assembled oligo construct should have complementary ends to the prepared vector
    • 1 μL 10X T4 DNA ligase buffer
    • H2O to 10 uL
  9. Add 0.5 μL T4 DNA ligase
  10. Incubate for 1hr 30 mins at 16°C.
  11. Transform.